CN113069590B

CN113069590B - Preparation method of regenerated bacterial cellulose composite hydrogel dressing

Info

Publication number: CN113069590B
Application number: CN202110228436.6A
Authority: CN
Inventors: 张继; 宋珅; 丁玲; 刘小媛; 黄玉龙; 杨生荣; 范增杰
Original assignee: Northwest Normal University
Current assignee: Northwest Normal University
Priority date: 2021-03-02
Filing date: 2021-03-02
Publication date: 2022-07-01
Anticipated expiration: 2041-03-02
Also published as: CN113069590A

Abstract

The invention discloses a preparation method of a regenerated bacterial cellulose composite hydrogel dressing, which is prepared by taking bacterial cellulose rich in reductive hydroxyl groups and polyvinyl alcohol as a dispersing agent and a reducing agent in a sodium hydroxide and urea green solvent, forming hydrogen bonds and reducing to generate nano silver at the same time, and performing repeated freeze thawing without using any toxic chemical cross-linking agent and reducing agent. The preparation method is simple and green, realizes the high-efficiency loading and slow release of the silver on the composite hydrogel, and overcomes the defect of high toxicity of chemical reducing agents and crosslinking agents. The hydrogel dressing has the advantages of low preparation cost, good mechanical property, excellent antibacterial property, biocompatibility and wound healing promotion performance, is an ideal wound dressing, and is expected to be applied to clinical skin wound treatment.

Description

Preparation method of regenerated bacterial cellulose composite hydrogel dressing

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a preparation method of a regenerated bacterial cellulose composite hydrogel dressing.

Background

The skin is the largest organ of the human body and plays an important role in maintaining homeostasis and preventing invasion of microorganisms. However, skin damage can lead to bacterial infection, tissue dehydration and severe secondary trauma. The hydrogel has the characteristics of high water content, good biocompatibility, porous three-dimensional structure and the like, so that the hydrogel becomes an ideal wound dressing for treating skin injury.

Hydrogels such as polyvinyl alcohol (PVA), Bacterial Cellulose (BC), and the like, have poor mechanical or antibacterial properties, limiting their application as wound dressings. The traditional method for enhancing the mechanical property of the hydrogel is to add an enhancing auxiliary material or use glutaraldehyde and a chemical cross-linking agent. The PVA/BC nano composite material is prepared by adopting a solution pouring method or a mechanical blending method and using BC as a reinforcing agent. In the process, although the cellulose can be uniformly distributed in the whole material, the BC can not be dissolved in a common solvent and can not be well combined with PVA to play a role in effectively improving the mechanical property, so that the mechanical property of the prepared hydrogel is poor. In order to further improve the mechanical properties, chemical cross-linking agents such as glutaraldehyde are used to improve the mechanical properties of the hydrogel. The nano silver (AgNPs) has excellent antibacterial activity and is widely applied to wound dressings. AgNPs are introduced into hydrogel, so that the antibacterial performance of the hydrogel can be improved, and the mechanical property of the hydrogel can be improved to a certain extent. However, although chemical reducing agents such as sodium borohydride can improve the preparation efficiency of silver nanoparticles, the chemical reducing agents generate potential biological toxicity on cells and tissues and are not beneficial to proliferation and growth of the cells and the tissues.

Disclosure of Invention

The invention aims to provide a preparation method of a regenerated bacterial cellulose composite hydrogel dressing.

Preparation of regenerated bacterial cellulose composite hydrogel dressing

The invention fully utilizes the characteristic that BC and PVA can improve the interaction with body cells to promote the migration of epithelial cells and fibroblasts, thereby accelerating the formation of tissues, takes AgNPs with good antibacterial performance as an antibacterial agent, overcomes the defects of complex preparation process, high preparation cost, high toxicity of chemical products such as a cross-linking agent and the like in the traditional antibacterial hydrogel preparation process, and provides a preparation method of a regenerated bacterial cellulose composite hydrogel dressing, which specifically comprises the following preparation steps:

(1) adding bacterial cellulose powder into NaOH, urea and H₂And (3) precooling the mixed solution for 30-60 min at the temperature of-12 to-20 ℃, and stirring for 10-30 min to obtain a transparent bacterial cellulose solution. Wherein the bacterial cellulose powder is mixed with NaOH, urea and H₂The mass ratio of the O mixed solution is 1: 25-1: 50; NaOH, Urea and H₂In O mixed solution, NaOH, urea and H₂The mass ratio of O is 1:1.5: 10-1: 2: 15; the stirring is carried out at 2000-3000 r/min.

The preparation method of the bacterial cellulose powder comprises the following steps: inoculating Acetobacter xylinum (ATCC 53582) into an HS culture medium, adjusting the pH to 5.5-6.5, and standing and fermenting for 7-10 days at the temperature of 25-30 ℃; and taking out the obtained upper layer bacterial cellulose film, placing the upper layer bacterial cellulose film in 0.1 mol/L NaOH solution, treating at 65-85 ℃ for 40-60 min, removing residual culture medium and thalli, taking out the bacterial cellulose film from NaOH, repeatedly washing with distilled water until the pH is =7.0, freeze-drying, mechanically crushing, and sieving with a 200-mesh sieve to obtain bacterial cellulose powder.

(2) Adding PVA into deionized water to obtain PVA solution, and adding AgNO into the PVA solution₃Mixing them uniformly and fully dissolving to obtain AgNO₃The PVA solution of (1). Wherein PVA and AgNO₃The mass ratio of (A) to (B) is 8: 1-50: 1.

(3) Mixing the bacterial cellulose solution obtained in the step (1) with AgNO obtained in the step (2)₃Mixing the PVA solution, stirring for 1-3 hours at 60-80 ℃, adding the mixed solution into a culture dish, repeatedly freezing and thawing to form hydrogel, washing the hydrogel with distilled water until the pH is =7.0, and obtaining the hydrogel which is the regenerated bacterial cellulose composite hydrogel dressing. Wherein, the bacterial cellulose solution and AgNO₃The volume ratio of the PVA solution is 1: 1-1: 2; the repeated freezing and thawing is firstly kept for 8-12 h at the temperature of-20 to-40 ℃, then kept for 3-5 h at room temperature, and the process is repeated for 3-4 times.

Second, the structure and performance of the regenerated bacterial cellulose composite hydrogel dressing

1. Structure of composite hydrogel dressing

The composite hydrogel dressing microstructure was characterized using SEM. FIG. 1a shows the microscopic morphology of the PVA/BC composite hydrogel without nanosilver, the interwoven fiber network structure of BC and PVA, forming uniform honeycomb cells with diameters of 1-2 μm. FIGS. 1 b, c and d show SEM images of regenerated bacterial cellulose composite hydrogel PVA/BC-Ag with different silver contents (b, c and d are respectively PVA/BC-Ag1, PVA/BC-Ag2 and PVA/BC-Ag3, and the scale bar in the images is 5 μm). Compared to BC/PVA hydrogels, the pore structure in the three-dimensional network structure of PVA/BC-Ag hydrogels contains more irregular saw-tooth and wedge-shaped structures, the surface becomes more irregular, and the pore size of the honeycomb structure increases to about 3-5 microns. These cellular structures may make these hydrogels more favorable for oxygen exchange, moisture retention, and cell adhesion and growth. FIGS. 1e-h show optical images of PVA/BC and PVA/BC-Ag hydrogels with different silver contents (e is PVA/BC, f-h is PVA/BC-Ag1, PVA/BC-Ag2, PVA/BC-Ag3, respectively), the hydrogel color deepens with increasing AgNPs content, and confirms that AgNPs has been successfully incorporated into the hydrogel.

2. Mechanical properties of composite hydrogel dressing

Good mechanical properties have an important influence on the application of hydrogels. In practice, the wound surface will inevitably accompany body movements, and therefore better mechanical properties, especially tensile properties, are required for wound dressings. The mechanical properties of the PVA/BC and PVA/BC-Ag hydrogels are shown in FIG. 2. Compared with PVA/BC, the PVA/BC-Ag hydrogel has better tensile strength. Furthermore, the mechanical properties of the composite hydrogel increased with increasing AgNP concentration. This is because the addition of AgNPs enhances the crosslinking effect between PVA and BC, thereby improving the mechanical properties of the hydrogel. The tensile strength and the elongation at break of the PVA/BC-Ag2 hydrogel reach 89.2 kPa and 215.6 percent respectively, and are respectively improved by 34.98 percent and 72.53 percent compared with the PVA/BC hydrogel. This result is also much greater than the tensile strength of PVA and the elongation at break of BC, so that these hydrogels can be prepared to fully meet the requirements of the wound dressing on the mechanical properties of the material.

3. Water absorption of composite hydrogel dressing

Wound dressings should have a high water absorption capacity to ensure that they absorb the fluids exuded from the wound surface in a timely manner. The BC, PVA/BC and PVA/BC-Ag hydrogels were immersed in the SBF solution for 24 hours to study their water absorption capacity. As shown in FIG. 3, the PVA/BC and PVA/BC-Ag hydrogels showed some water absorption properties after the addition of PVA. Compared with PVA/BC, PVA/BC-Ag hydrogel has better water absorption performance than PVA/BC hydrogel, because AgNPs enhance the crosslinking effect, BC/PVA-AgNPs hydrogel formed by crosslinking has larger pore diameter and gaps than BC/PVA hydrogel 3D network structure, and has a looser structure and stronger water absorption capacity. The water absorption swelling rate of PVA/BC-Ag3 reaches 1604.9 +/-58.2 percent, which is obviously higher than that of PVA and chitosan. Therefore, the high swelling ratio of PVA/BC-Ag can ensure the efficient absorption of the exudate, and completely meets the requirements of the wound dressing.

4. Antibacterial property of composite hydrogel dressing

The antimicrobial activity of the hydrogel was studied using the most common skin infection microorganisms (E.coli and Staphylococcus aureus), and the growth of the bacteria was observed by plate counting. As shown in FIG. 4, the PVA/BC hydrogel has no antibacterial activity, while the antibacterial ability of the PVA/BC-Ag hydrogel is significantly increased with the increase of the AgNPs content. The sterilization rates of PVA/BC-Ag1 on Escherichia coli and Staphylococcus aureus are 65.63 +/-2.63% and 51.17 +/-1.49% respectively, and the sterilization rates of PVA/BC-Ag3 on Escherichia coli and Staphylococcus aureus are 99.72 +/-0.14% and 99.38 +/-0.48% respectively (FIG. 5).

In conclusion, BC is dissolved in a sodium hydroxide/urea green solvent system, bacterial cellulose rich in reducing hydroxyl groups and polyvinyl alcohol are used as a dispersing agent and a reducing agent to form hydrogen bonds and reduce to generate nano silver, and the regenerated bacterial cellulose composite hydrogel dressing is prepared by a repeated freeze-thaw method under the condition that no toxic chemical cross-linking agent or reducing agent is used. The preparation method is simple and green, realizes the high-efficiency loading and slow release of the silver on the composite hydrogel, and overcomes the defect of high toxicity of chemical reducing agents and crosslinking agents. The hydrogel dressing has the advantages of low preparation cost, good mechanical property, excellent antibacterial property, biocompatibility and wound healing promotion performance, is an ideal wound dressing, and is expected to be applied to clinical skin wound treatment.

Drawings

FIG. 1 is SEM images (a-d) and photomicrographs (e-h) of a composite hydrogel dressing;

FIG. 2 shows the mechanical properties of PVA/BC and PVA/BC-Ag composite hydrogels;

FIG. 3 shows the water absorption of PVA/BC and PVA/BC-Ag composite hydrogels;

FIG. 4 shows the growth of the BC/PVA and BC/PVA-AgNPs composite hydrogel on an agar plate after incubation with Escherichia coli and Staphylococcus aureus;

FIG. 5 shows the bactericidal rate of the BC/PVA and BC/PVA-AgNPs composite hydrogel.

Detailed Description

The following provides a further explanation of the preparation method of the regenerative bacterial cellulose composite hydrogel dressing according to the present invention by using specific examples.

Example 1

(1) Acetobacter xylinum (ATCC 53582) was inoculated into HS medium, adjusted to pH 5.5, and left to ferment at 26 ℃ for 7 days. Taking out the upper layer Bacterial Cellulose (BC) film, placing in 0.1 mol/L NaOH solution, treating at 70 deg.C for 40min, and removing residual culture medium, thallus and other impurities. After the BC membrane was removed from NaOH, the membrane was repeatedly washed with distilled water to PH =7.0 to remove NaOH remaining in the membrane. Freeze drying, mechanically pulverizing, and sieving with 200 mesh sieve to obtain BC powder.

(2) 2.0g of BC powder was slowly added to 48 g of NaOH, urea and H₂O is mixed (7:12:81, w/w) in the solution, precooled for 30min at-12 ℃ and rapidly and mechanically stirred for 10 min at 2000 r/min to prepare transparent BC solution.

(3) Adding 5 g PVA into 45 ml deionized water, mechanically stirring at 85 deg.C for 2 hr to dissolve it sufficiently, adding 2 mM AgNO₃Adding PVA solution to mix and dissolve to obtain AgNO-containing solution₃The PVA solution of (1).

(4) Mixing the BC solution with AgNO₃The PVA solution is 1:1(v/v) mixing and stirring at 60 ℃ for 1 h. And adding the mixed solution into a culture dish, repeatedly freezing and thawing for 3 times (firstly at-20 ℃ for 8 h, and then at room temperature for 3 h) to form hydrogel, washing the hydrogel with distilled water until the pH is =7.0, and removing unreacted substances to obtain the hydrogel, namely the regenerated bacterial cellulose composite hydrogel dressing PVA/BC-Ag 1.

The elongation at break of the tensile strength of PVA/BC-Ag1 was 70.1 KPa and 168.5%, respectively, the water absorption rate was 1390.4%, and the bactericidal rates against Escherichia coli and Staphylococcus aureus were 65.7% and 51.2%, respectively.

Example 2

(1) Acetobacter xylinum (ATCC 53582) was inoculated into HS medium, adjusted to pH 6.0, and left to ferment at 28 ℃ for 8 days. Taking out the upper layer Bacterial Cellulose (BC) film, placing in 0.1 mol/L NaOH solution, treating at 75 deg.C for 50 min, and removing residual culture medium, thallus and other impurities. After the BC membrane was removed from NaOH, the membrane was repeatedly washed with distilled water to PH =7.0 to remove NaOH remaining in the membrane. Freeze drying, mechanically pulverizing, and sieving with 200 mesh sieve to obtain BC powder.

(2) 2.0g of BC powder was slowly added to 48 g of NaOH, urea and H₂O mixed (7:12:81, w/w) in the solution, precooling for 40min at-15 ℃, and rapidly and mechanically stirring for 20 min at 2500 r/min to prepare a transparent BC solution.

(3) Adding 5 g PVA into 45 ml deionized water, mechanically stirring at 90 deg.C for 3 hr to dissolve it sufficiently, adding 3mM AgNO₃Adding PVA solution to mix and dissolve to obtain AgNO-containing solution₃The PVA solution of (1).

(4) Mixing the BC solution with AgNO₃The PVA solution (A) is mixed evenly according to the ratio of 1:1 (V/V), and stirred for 2 hours at the temperature of 70 ℃. And adding the mixed solution into a culture dish, repeatedly freezing and thawing for 4 times (firstly at minus 30 ℃ for 10 h, and then at room temperature for 4 h) to form hydrogel, washing the hydrogel with distilled water until the pH is =7.0, and removing unreacted substances to obtain the hydrogel, namely the regenerated bacterial cellulose composite hydrogel dressing PVA/BC-Ag 2.

The elongation at break of the tensile strength of PVA/BC-Ag2 was 89.2 KPa and 215.6%, respectively, the water absorption rate was 1440.3%, and the bactericidal rates against Escherichia coli and Staphylococcus aureus were 87.4% and 92.5%, respectively.

Example 3

(1) Acetobacter xylinum (ATCC 53582) was inoculated into HS medium, adjusted to pH 6.5, and left to ferment at 30 ℃ for 10 days. Taking out the upper layer Bacterial Cellulose (BC) film, placing in 0.1 mol/L NaOH solution, treating at 85 deg.C for 60min, and removing residual culture medium, thallus and other impurities. After the BC membrane was removed from NaOH, the membrane was repeatedly washed with distilled water to PH =7.0 to remove NaOH remaining in the membrane. Freeze drying, mechanically pulverizing, and sieving with 200 mesh sieve to obtain BC powder.

(2) 2.0g of BC powder was slowly added to 48 g of NaOH, urea and H₂O mixed (7:12:81, w/w) in the solution, precooling for 60min at-20 ℃, and rapidly and mechanically stirring for 30min at 3000r/min to prepare a transparent BC solution.

(3) Adding 5 g PVA into 45 ml deionized water, mechanically stirring at 95 deg.C for 4 hr to dissolve it sufficiently, adding 5 mM AgNO₃Adding PVA solution to mix and dissolve to obtain AgNO-containing solution₃The PVA solution of (1).

(4) Mixing the BC solution with AgNO₃The PVA solution (B) is mixed evenly according to the ratio of 1:1 (V/V) and stirred for 3 hours at the temperature of 80 ℃. And adding the mixed solution into a culture dish, repeatedly freezing and thawing for 3 times (firstly at minus 40 ℃ for 12 hours, and then at room temperature for 5 hours) to form hydrogel, washing the hydrogel with distilled water until the pH is =7.0, and removing unreacted substances to obtain the hydrogel, namely the regenerated bacterial cellulose composite hydrogel dressing PVA/BC-Ag 3.

The elongation at break of the tensile strength of PVA/BC-Ag3 was 73.3 KPa and 179.2%, respectively, the water absorption rate was 1604.9%, and the bactericidal rates against Escherichia coli and Staphylococcus aureus were 99.7% and 99.4%, respectively.

Claims

1. A preparation method of a regenerated bacterial cellulose composite hydrogel dressing comprises the following steps:

(1) adding bacterial cellulose powder into NaOH, urea and H₂Precooling for 30-60 min at-12 to-20 ℃ in O mixed solutionStirring for 10-30 min to obtain a transparent bacterial cellulose solution;

(2) adding PVA into deionized water to obtain PVA solution, and adding AgNO into the PVA solution₃Mixing them uniformly and fully dissolving to obtain AgNO₃The PVA solution of (4);

(3) mixing the bacterial cellulose solution obtained in the step (1) with AgNO obtained in the step (2)₃Mixing the PVA solution, stirring for 1-3 hours at 60-80 ℃, adding the mixed solution into a culture dish, repeatedly freezing and thawing to form hydrogel, washing the hydrogel with distilled water until the pH is =7.0, and obtaining the hydrogel which is the regenerated bacterial cellulose composite hydrogel dressing.

2. The preparation method of the regenerative bacterial cellulose composite hydrogel dressing as claimed in claim 1, wherein the preparation method comprises the following steps: in the step (1), the preparation method of the bacterial cellulose powder comprises the following steps: inoculating acetobacter xylinum into an HS culture medium, adjusting the pH value to 5.5-6.5, and standing and fermenting for 7-10 days at the temperature of 25-30 ℃; and taking out the obtained upper layer bacterial cellulose film, placing the upper layer bacterial cellulose film in 0.1 mol/L NaOH solution, treating at 65-85 ℃ for 40-60 min, removing residual culture medium and thalli, taking out the bacterial cellulose film from NaOH, repeatedly washing with distilled water until the pH is =7.0, freeze-drying, mechanically crushing, and sieving with a 200-mesh sieve to obtain bacterial cellulose powder.

3. The preparation method of the regenerative bacterial cellulose composite hydrogel dressing as claimed in claim 1, wherein the preparation method comprises the following steps: in the step (1), bacterial cellulose powder is mixed with NaOH, urea and H₂The mass ratio of the O mixed solution is 1: 25-1: 50.

4. The preparation method of the regenerative bacterial cellulose composite hydrogel dressing as claimed in claim 1, wherein the preparation method comprises the following steps: in step (1), NaOH, urea and H₂In O mixed solution, NaOH, urea and H₂The mass ratio of O is 1:1.5: 10-1: 2: 15.

5. The preparation method of the regenerative bacterial cellulose composite hydrogel dressing as claimed in claim 1, wherein the preparation method comprises the following steps: in the step (1), stirring is carried out at 2000-3000 r/min.

6. The preparation method of the regenerative bacterial cellulose composite hydrogel dressing as claimed in claim 1, wherein the preparation method comprises the following steps: in step (2), PVA and AgNO₃The mass ratio of (A) to (B) is 8: 1-50: 1.

7. The preparation method of the regenerative bacterial cellulose composite hydrogel dressing as claimed in claim 1, wherein the preparation method comprises the following steps: in the step (3), the bacterial cellulose solution and AgNO₃The volume ratio of the PVA solution (b) is 1: 1-1: 2.

8. The preparation method of the regenerative bacterial cellulose composite hydrogel dressing as claimed in claim 1, wherein the preparation method comprises the following steps: in the step (3), the repeated freezing and thawing is firstly kept for 8-12 hours at the temperature of-20 to-40 ℃, then kept for 3-5 hours at the room temperature, and the process is repeated for 3-4 times.